Blood separation device comprising a filter and a capillary flow pathway exiting the filter

ABSTRACT

A method for separating plasma from red blood cells and a device utilizing the method in which a low-pressure filter is interposed in a pathway between an inlet port and a reaction area. The sole driving force for the movement of plasma from the filter to the reaction area is capillary force provided by a tubular capillary. The filter is selected from glass microfiber filters of specified characteristics, which can operate in the absence of agglutinins, and filters capable of separating agglutinated red cells from a plasma, which require the use of an agglutinin.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to techniques and devices for separating plasmafrom blood by filtration and is particularly directed to filtration atlow pressures.

2. Description of the Background

Many diagnostics are carried out in the clinical field utilizing bloodas a sample. Although some of these techniques can be carried out onwhole blood, it is necessary in many instances to utilize serum orplasma as the sample in order to obtain an accurate reading. Forexample, red blood cells (erythrocytes) scatter and absorb light andcould adversely affect a measurement of either reflected or transmittedlight of a diagnostic test relying on either of these measurementtechniques.

Traditionally, plasma and serum have been separated from whole blood bycentrifuging either before (for plasma) or after (for serum) clotting.However, centrifugation is time consuming and requires equipment that isnot generally available outside the clinical laboratory. Accordingly,field testing of numerous blood substances that require serum or plasmais difficult.

A number of techniques have been devised to avoid this problem. Thetechniques generally utilize a filtering device capable of separatingred blood cells from plasma. Numerous materials have been used in thepast to form filters. Paper, non-woven fabric, sheet-like filtermaterial composed of powders or fibers such as man-made fibers or glassfibers, and membrane filters having suitable pore sizes have beenproposed For example, U.S. Pat. No. 4,256,693 to Kondo et al. disclosesa number of filter materials in a multi-layered integral chemicalanalysis element for use with blood. U.S. Pat. No. 4,477,575 to Vogel etal. describes a composition and process for permitting the separation ofplasma or serum from whole blood utilizing glass fibers in combinationwith other absorbent layers.

However, these prior art techniques have proven to be unsuitable for usein applications which, because of space and volume restraints, can onlyutilize a small filter in a device in which a single drop of blood isseparated and the plasma is transported through the device solely bymeans of capillary action. Accordingly, further refinement in bloodseparation techniques is desirable.

SUMMARY OF THE INVENTION

A device and a technique for separating red blood cells from plasma areprovided in which a whole blood sample is applied to a filter underconditions in which the driving force for transporting the plasma fromthe exit face of the filter is provided solely by capillary action. Twobasic filtering techniques can be used. The first utilizes a glassmicrofiber filter and does not require the use of red cell agglutinins(although an agglutinin can be used if desired). The second requires theuse of agglutinins but can employ a wide variety of filters.

The glass microfiber filter is selected in terms of particle sizeretention and thickness to allow plasma to pass more rapidly through thefilter than the red blood cells, whose passage through the filter isretarded in a manner similar to that which occurs in chromatographycolumns. Although the red blood cells eventually pass through thefilter, sufficient plasma has separated and passes by capillary actionto a reaction chamber to allow analysis of the analyte present in theplasma without interference by the red blood cells. When agglutinins areused, the filter can be any filter capable of separating agglutinatedred blood cells from plasma. However, both techniques are speciallyadapted for use with small volumes of blood and the low pressuresavailable for use in transporting blood in capillary devices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows one embodiment of a filter-containing device of theinvention in which a number of examples described below were carried outin which

FIG. 1a is an expanded side view, FIG. 1b is a bottom view of each ofthe components making up the final device, and FIG. 1c is a top view ofthe assembled device.

FIG. 2 shows an embodiment of a filter-containing device of theinvention in which two or more plastic forms are welded to form aunitary device having internal chambers in which FIG. 2a is a side viewand FIG. 2b is a top view of the unitary device after welding.

FIG. 3 is a top view of a filter-containing device of the inventionhaving multiple pathways for the passage of separated plasma to areagent chamber.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention may be carried out in the capillary flow devicethat is described in detail in U.S. application Ser. No. 880,793, filedJuly 1, 1986, which is a Continuation-In-Part of U.S. application Ser.No. 762,748, filed Aug. 5, 1985, both of which are commonly assigned,co-pending applications. The capillary flow device described in theseearlier applications relies upon capillaries, chambers, and orifices topump fluids: to control measurement of fluids, reaction times, andmixing of reagents: and to determine a detectable signal. Thecapillaries provide the sole driving force for the movement of liquidthrough the device.

Although these devices could be utilized with whole blood as previouslydescribed, use with serum or plasma required separation of red bloodcells prior to application of the serum or plasma to the device. Thepresent invention allows application of whole blood directly to thesedevices or to any other devices which rely on capillary action toprovide the driving force for the movement of fluids. By selecting glassfiber filters or combinations of agglutinins and either glass ornon-glass filters as described in this specification, it is possible toaccomplish the desired separation in a very small space with a minimumof cell lysis and without requiring the application of any additionalforce other than that which is supplied by capillary action to move theserum or plasma to a reaction chamber.

One useful aspect of the invention is that separation of red blood cellsfrom plasma can be accomplished utilizing a single layer of filtermaterial and a small volume of blood. Prior art materials used for bloodseparation on a larger scale and/or utilizing multiple-layer filterswith absorbent layers have proven not to be useful under the presentconditions for separation.

A key part of a first embodiment of the present device is a glass fiberfilter. Particularly suitable glass fiber filters can be prepared fromfibers of borosilicate glass, a material that contains, in addition tosilicon dioxide, approximately 10% of boron trioxide as well as alkaliand alkaline earth oxides and oxides of other metals such as iron,aluminum, and zinc. However, other glasses can also be utilized.

In the production of glass fiber filtering media of the invention,microglass fibers are utilized. These are extremely fine fiberstypically formed by blowing glass through jets as opposed to spun glassmaterial made from drawn glass filaments. Typically, glass fiber filtersare prepared from fibers with diameters between 0.10 and 7.0 μm.

However, it is important to control the distribution of fibers presentwithin this diameter range in order to prepare a glass fiber filter thatwill be useful in the practice of this invention. A narrow range of finefibers with a minimum of large diameter fibers should be used.

A preferred filter will have 60%, preferably 80% or more, of its fiberswith diameters from 0.10 to 1.23 μm and no more than 40%, perferably nomore than 20%, with diameters larger than 1.23 μm. Filters withessentially all of their fibers having diameters less than 4.00 μm arepreferred.

On the other hand, the range of fiber sizes should not be too smallwithin the limits outlined above. A relatively even distribution ofdiameters in the range of 0.10 to 1.23 μm is preferred. An extremelynarrow range of fiber diameters (varying over a total range of 0.14 μm)has been shown to be incapable of providing correct filter action.Accordingly, it is preferred to utilize a distribution of fibers ofdifferent diameters so that if the 0.10 to 1.23 μm range is divided into2-5 equal divisions, especially 3 or 4 equal divisions, approximatelyequal numbers of fibers (preferably varying by no more than 10 numberpercent) will fall into each division (e.g. a 40, 30, 30: 30,40,30: or35, 30, 35 number ratio upon division into three ranges of diameter).

Suitable filter sheets can be prepared by applying a mixture of glassfibers in a wet pulp in a paper-making machine. In some cases, a smallamount of a high-polymer organic binder can be utilized although suchbinders are not preferred. Typical binders include cellulosic or acrylicpolymers.

The glass fiber filters used in the practice of the invention are knownas depth filters, being composed of irregularly filtering fibers.Separation is obtained mainly as a result of mechanical retention ofparticles. Because of both the irregular size and shape of the fibers,it is difficult to give an absolute pore size in such a filter. Thefilters are generally classified based on retention, which defines thecapacity of a filter to remove particles of a given size from an aqueousor other solution.

In selecting glass filters, particle size retention, composition ofglass thickness, and density should be taken into consideration in orderto provide adequate filtration without hemolysis. A thickness of from0.5 to 0.9 mm is preferred, with 0.50 to 0.80 being more preferred,particularly from 0.66 to 0.76 mm. Borosilicate and other glass that isslightly alkaline (pH 8.0-11.0, preferably about 9.0-10.5) is preferred.Particle size retention is preferably from about 1.0 to 3.0 microns,more preferably from 1.4 to 3.0 microns, and most preferably from 2.3 to3.0 microns. A density in the range of from 0.10 to 0.30 g/cm³ ispreferred, more preferably 0.20 g/cm³ to 0.28 g/cm³, and most preferablyabout 0.25 g/cm³. Since the approximate density of borosilicate glass is2.61 g/cm³, density can be seen to be a measure of the porosity of theglass filter.

The numbers set forth above are given for borosilicate glass filters.Particle size retention and thicknesses would be the same for othertypes of glass, although the densities would vary proporationately withthe density of the respective glass selected.

A number of commercially prepared glass filters can be utilized in thepractice of the invention. For example, Micro Filtration Systems (MFS)manufactures three glass fiber filters that can be utilized, identifiedby the manufacturing numbers GA-200, GB-100R and GC-90. GB-100R andGC-90 are utilized as doubled filters in the practice of the presentinvention. GA-200 has a density of approximately 0.25 g/cm³, a thicknessof 0.70 mm, and a retention size of 2.3 microns when filtering liquids.A double thickness of GB-100R has a density of 0.25 g/cm³, a thicknessof 0.76 mm, and a particle size retention of 2.0 micron. A doubled layerof GC-90 has a density of 0.30 g/cm³, a thickness of 0.66 mm, and aparticle size retention of 1.7 micron.

Whatman, Inc., of Clifton, N.J., and Schleicher & Schuell, a West Germanfirm with a distribution in Keene, N.H., also manufacture a number ofdifferent glass microfiber filters. However, none of the Whatman orSchleicher & Schuell filters tested (Whatman GF/C, GF/B, GF/D, GF/F,934-4H: S+S 3362) has proven to be useful for the purpose of thisinvention, because of a difference in distribution of sizes of the glassfibers used to manufacture their filters and the resulting effects onred blood cell retention. Other glass fiber filters have also beentested and have been demonstrated not to provide adequate separation:P300, from Nucleopore, Pleasanton, Calif. (with organic binder); HB-5341and BG-08005, from Hollingsworth & Vose, East Walpole, Mass.: glassfiber filter 111, 121, 131, 141, 151, and 161, from Eaton-Dikeman,Carlisle, Pa.: and glass fiber filters 85/90F, from by Machery & Nagel,Duren, West Germany.

All of the manufactured glass fibers described above (except wherenoted) are prepared without organic binders. Organic binders tend toreduce pore sizes and otherwise interact with red blood cells as theypass through filters. Accordingly, binderless glass filters arepreferred. However, it may be possible to utilize binders in glassfilters by selecting densities and fibers sizes that result in equalparticle size retention. Furthermore, the strict control described doesnot need to be maintained when utilizing an agglutinin, as describedbelow.

A number of different filter types were tested for their ability toeffect the separation of plasma from serum using a device whose onlymotive force is capillary action. Of all the filters tested, binderlessglass fiber filters having the distribution of fiber diameters discussedabove gave the best separation. The pressure differential caused bycapillary action is apparently significantly lower than that whichexists either as a result of the action of gravity on larger samples oras a result of contact of a glass filter of the type described in U.S.Pat. No. 4,477,575, discussed above, with an absorbant pad. Typically,the available pressure is on the order of 2.5 mmHg (34 mm H₂ O) or less.

Binderless glass microfiber filters having a volume of approximately7-10 μl yielded about 3-4 μl of plasma when 25 μl of blood was applied.When the filter was utilized in a device as shown in FIG. 1, which isdescribed in detail below, plasma appeared at the top of the filteroutlet about five seconds after application of whole blood to thefilter. Plasma appeared in the well about twelve seconds afterapplication. Although blood cells eventually came through the filter,indicating that the blood cells were not being blocked but were beingretarded, sufficient plasma had appeared by this time in order toconduct an adequate analysis. Filters of this type have been shown to beuseful in filtering blood with hematocrits ranging from 33 to 60%. Theratio of plasma obtained to filter volume can be increased by utilizinglarger diameter filter while maintaining the same filter thickness.

It is also possible to separate plasma from red blood cells in a singledrop of blood in a capillary flow device using antibodies to red bloodcells or other agglutinins in combination with a filter. The filter canbe either the glass fiber filters described above (including the filtersthat do not work in the absence of agglutinins), paper, or any othertype of filter that can filter agglutinated red blood cells. Paper,non-woven fabrics, sheet-like filter material composed of powders orfibers (such as carbon or glass fibers), and membranes having suitablepore sizes can all be utilized with antibodies and other agglutinins.Cellulose fibers, cotton linters, nitrocellulose, wood pulp,α-cellulose, cellulose nitrate, and cellulose acetate are all suitablefor manufacturing acceptable filters and/or membranes.

Agglutinins can be present in the filter (in soluble form) or can beadded to the blood sample prior to filtering (for example, by having awhole blood sample pass through a capillary or other chamber containingsoluble agglutinins prior to contacting the filter). Any chemical orbiochemical agent capable of causing agglutination of red blood cell canbe used, including but not limited to antibodies and lectins. Suchagglutinins are well known in the field of chemical analysis. Antibodiesare preferred agglutinins, particularly for use with undiluted wholeblood. However, other soluble agglutinins are also satisfactory, bothfor direct and indirect agglutination of red blood cells. See, forexample, Stites et al., Basic and Clinical Immunology, 4th ed., LangeMedical Publications, Los Altos, Calif., (1982), pp 356-359.

The antibodies utilized will have binding affinity for a determinantpresent on the surface of red blood cells. If a specific monoclonalantibody that reacts with a blood antigen is used, such as an antibodythat reacts with type-A antigen, it will be necessary to match the bloodtype to the filter being used. Antibodies reactive with any antigenpresent on the surface of a red blood cell can be utilized, includingbut not limited to major histocompatability antigens, cell surfaceproteins, cell surface carbohydrates, and cell surface glycoproteins.

It is preferred to utilize a source of mixed antibodies that will reactwith all red blood cells of the species being tested. For example, anantiserum against human red blood cells can be utilized or a mixture ofmonoclonal antibodies that react with all of the major blood types. Suchantibodies are available commercially. For example, an IgG fraction ofrabbit anti-human red blood cell antibodies can be obtained from CooperBiomedical (Westchester, Pa.). The antibody can be adsorbed onto thesurface of the solid used to prepare the filter. In the case of paperfilters, antibody can be effectively adsorbed onto paper by merelycontacting the paper with an aqueous solution containing the antibodyand then removing the water by evaporation. If desired, an antiserum canbe applied neat or it may be diluted. There is generally a minimumamount of antibody that must be applied to the filter in order forfiltration to be effective. If less than the minimum amount is present,red blood cells pass too quickly through the filter. However, it is notpossible to give a specific amount of an antiserum that must be appliedto the filter since different antisera will differ in their ability tobind red blood cells. Accordingly, the optimum amount of antibody isdetermined empirically. Serial two-fold dilutions of neatantibody-containing solution or antiserum are applied to filters in anamount sufficient to saturate the filter. Efficiency of filtration,lysis of red blood cells, and amount of plasma that passes through thefilter when a standard amount of whole blood is applied are measured.When the IgG fraction of rabbit anti-human red blood cell antibody fromCooper Biomedical was utilized, the solution was reconstituted to give30 mg/ml of protein and 20 mM phosphate-buffered saline at a pH of 7.3.The minimum volume of this solution that appeared to be necessary forgood filtration was 7.5 μl (filter diameter 0.18 inch utilizing S+SGB003 paper; the filter volume was approximately 10 μl). However, it wasnot necessary to apply the antibody as a neat solution. Dilutions of1:10 were still effective in providing efficient filtration.Accordingly, it appears that the volume of solution (10 μl in a 1:10dilution) necessary to saturate the filter is more important thanproviding a high titer of antibody. When using a filter paper disk 0.180inch in diameter and a volume of approximately 10 μl, at least 5 μl,preferably at least 7.5 μl of solution appeared to be necessary tosaturate the disk and uniformly distribute the antibody throughout thefilter. Similar volume ratios (0.5:1 and 0.75:1) will be effective forother filter volumes. Uniform distribution of antibody prevents redblood cells from passing through the filter at one location while beingtrapped in others.

If antibody is added to the sample prior to contact with the filter, itis preferred to carry out the filtration in the presence of an agentcapable of suppressing hemolysis. Typical suppressing agents includelocal anaesthetics, such as dibucaine and lidocaine: β-andrenergicblockers, such as propanolol: tricyclic antidepressants, such aschlorpromazine and anitriptreine: and 3-hydroxypyridines, such as3-hydroxy-6-methylpyridine.

It may be possible to utilize a filter, with or without antibody, tocontrol the rate of passage of plasma or blood (the latter whenutilizing a bare paper filter or other material that does not separatered blood cells from plasma). Increasing the amount of antibody on afilter increases the time that it takes the plasma front to reach agiven location along the capillary path. The filter and the capillaryleaving the filter each act as a point of resistance to the flow offluid through the device. In effect, each acts as a valve in a fluidstream. When passage of fluid through the filter meets with moreresistance than flow through the capillary, the system acts as if afirst valve is partially closed while a second valve in the fluid streamis open. However, it is possible to vary the capillary flow rate so thatgreater resistance is present in the capillary. Such a system acts as ifthe first valve is open while the second valve is partially closed. Byvarying filter thickness and density and by selecting an appropriatecapillary diameter, considerable control over flow of fluid through thesystem can be achieved.

The filter as described above has been utilized in the test devicesdescribed in U.S. patent application Ser. Nos. 880,793 and 762,748,which are herein incorporated by reference. A brief description of thesedevices is included here to show how the filter is used in combinationwith the remainder of a device that utilizes (1) small volumes of bloodand (2) capillary action to cause movement of plasma.

A test device utilized in many of the experimental investigationsdescribed below is set forth in FIG. 1. The device was prepared fromthree plastic pieces approximately the size and shape of microscopeslides and double-sided tape. Top slide 10 had a hole 12 smaller indiameter than the filter to be utilized drilled completely through slide10 and double-sided tape 14, which in the embodiment shown does notextend the full length of the top slide but may do so if desired. Middleslide 20 has a hole 22 drilled completely through slide 20 anddouble-sided tape 24, which is applied to the bottom surface of slide20. Double-sided tape 24 has a section 26 cut out of the tape to providecapillary channels and chambers when the total device is assembled.Capillary space 26A leads from hole 22, which holds the filter, toreaction chamber 26B. An additional capillary chamber 26C provides avent by extending from the reaction chamber to the edge of the tape.Bottom slide 30 is a plain slide that forms a bottom surface of thefilter, capillary, and reagent spaces formed by middle slide 20 and tape24.

The assembled device is shown in FIG. 1C in which dotted lines areutilized to show the internal chambers that have been formed. Blood isapplied at entry port (hole) 12, contacts the filter held in chamber 22,and is separated into plasma while the red blood cells are retained onthe filter. Plasma passes through capillary 26A to reaction chamber 26Bwhile air is vented through capillary vent 26C.

FIG. 2 shows a device prepared by welding two or more plastic piecestogether to form a unitary device having internal chambers. Numerousembodiments of this device are set forth in U.S. patent application Ser.Nos. 880,793 and 762,748, referenced above. Blood is applied to entryport 42, which is smaller in diameter than chamber 44 which containsfilter 46. Plasma exits the bottom of the filter into collecting space48 and is transported by capillary 50 to reaction chamber 52. Vent 54 isprovided for exit of air from the device. Ridges 56 may be provided ifdesired to aid in the application of blood to the entry port. Additionalcapillaries, chambers, vents, and the like such as are described in theincorporated patent applications may be present in device 40 but areommitted in FIG. 2 for clarity.

A whole blood sample, optionally formulated by addition ofanticoagulants or other reagents useful in collection of blood or inundergoing a reaction with the analyte that will be measured, isintroduced into the entry port in the receiving unit of a test device.The receiving unit may be a capillary or a larger chamber. The receivingunit may be used to measure the particular sample volume or may simplyserve to receive the sample and direct the sample to the filter. Whenwhole blood contacts the filter, it is separated into its components asdescribed above. The first component to leave the filter will be plasmaor serum, depending on the source of the sample. For the remainder ofthis discussion the term plasma will be used but should be understood torepresent either plasma or serum.

The filters of the present invention typically comprise a single layerof material rather than multiple layers. They are intended forseparation of a single drop of blood, which typically has a volume of30-50 μl or less. Accordingly, the volume of the filter is also small,typically in the range of 5 to 20 μl, in order to avoid absorbing andretaining all of the plasma. Thickness (i.e., measured in the directionof the flow path) is preferably in the range of 0.2-1.5 mm. This rangeis for all filters and thus is somewhat broader than that expressed forglass microfiber filters set forth above. Particle size retention forglass microfiber filters is discussed above. Filters used withagglutinins can be more porous if desired but should retain agglutinatedred blood cells, which typically form clumps of cells with apparentdiameters from 6-10 μm for a few cells to greater than 0.1 mm (100 μm)for a large number of cells.

The plasma will usually be picked up as it leaves the filter by one ormore capillaries. When blood is applied to the top of a filter, plasmawill be collected from the bottom. The sides of the filter are in closecontact with the walls to prevent red blood cells from passing aroundthe edges of the filter. Optionally, a sealer (usually a polymericcompound) can be used on the sides of the filter. Plasma leaving thebottom of the filter can collect in grooves or other spaces between thefilter and the surface of the device containing the filter in closestcontact with the bottom of the filter. Capillaries will draw plasma offfrom the collection space or spaces. It will be recognized that thewords top, bottom, and sides as used here are relative terms and do notnecessarily describe orientation of the filter in relation to theearth's surface. Capillaries will usually have diameters in the range ofabout 0.01 mm to 2 mm. The capillaries will vary in length but aregenerally shorter than 10 cm. usually not exceeding about 5 cm.

The first capillary may control the rate of flow into the chamber thatwill usually serve as the reaction chamber. Thus, the capillary may aidin the control of the time with which the plasma is in contact with areagent contained within or bound to the walls of the capillary and/orreaction chamber. However, the flow rate of plasma through the filter islimiting in many instances, as described above, so that the capillaryoften is transporting plasma as fast as it leaves the filter. Thereagent provides a color change or some other means of determining theamount of analyte present in the plasma.

The capillary provides the sole driving force for the movement of liquidthrough the device after passage of the sample through the filter. Thedevice is normally employed with the capillaries, reaction chambers, andother chambers being oriented in a horizontal plane so that gravity doesnot affect the flow rate. The device is employed without ancillarymotive force, such as a pump, gravity, or the like. Accordingly, it isessential to select a filter as described herein in order to achieve theseparation while allowing capillary force to transport plasma throughthe device. Experimental evidence has demonstrated that the filtersdescribed in prior art such as U.S. Pat. Nos. 4,477,575 and 4,256,693,for separating large volumes of blood aided by gravity or which dependon relatively large wicking forces caused by absorbant substances thatcontact the filter, are ineffective in capillary flow devices of thetype utilized in the present invention.

Although the filters described herein can be utilized in the samedevices previously described, a preferred configuration for use ofdevices with glass fiber filters is shown in FIG. 3. In this device,whole blood is supplied to an entry port 42' situated above a filter,designated as a blood separater. A number of capillaries (50') arearranged at the periphery of the blood separater to transport plasma tothe reagent area. The capillaries may be of different lengths anddiameters but are designed to allow plasma to reach the reagent area 52'substantially simultaneously from each capillary. U.S. application Ser.No. 880,793 describes sizing capillaries to achieve this affect. Thisdesign allows for uniform and rapid filling of the reagent area.

The invention will now be further described by reference to certainspecific examples which are included for purposes of illustration onlyand are not to be considered limiting of the invention unless otherwisespecified.

EXAMPLE I Materials and Methods

Blood. Whole blood in 15 USP units/ml of lithium heparin was used in thefollowing experiments.

Filter disks. The filter disks were made from commercialy availablefilters or other indicated materials by using a 0.180" punch.

Welded Cartridges. ABS (acrylamide butadiene styrene) slides were weldedwith the Branson ultrasonic welder at the following settings:pressure=60 psi, weld time=0.3 sec, hold time=1.5 sec, down speed=3.0.

The essential parts of the device were a filter chamber 33.5 mil thickwith a total volume of 16 μl, a connecting chamber (wider than a normalcapillary) 3.5 mm thick, and a reaction chamber with vent hole. Thetotal volume of the connecting chamber and reaction chamber was 8.5 μl.

Tape Slides. Acetate plastic strips (6"×1") were washed in Sparkleensolution, rinsed in deionized water, and then dried using lint freetowels. The plastic strips were then cut into 2.5"×1" slides. Plasticsurfaces that contacted plasma were etched in a plasma etcher prior toassembly. The top slide was a clean piece of plastic with a 1"×0.5"double stick tape piece stuck to the bottom of the slide. Adouble-sided, 3.5 mil thick, Scotch brand tape with a pattern thatformed capillaries and other internal chambers cut out of the tape wasstuck to the bottom of what would be the middle slide. A hole wasdrilled to form the well using a #16 drill (0.173"). A #25 drill wasused to make a vent hole in this cover slide. The top strip was stuck tothe top of the middle strip with the holes carefully aligned. The filterof choice is then placed in the well of the middle slide, and a bottometched slide was stuck to the middle slide's tape. The filter was flushagainst the top surface of the bottom slide. The finished slide is shownin FIG. 1.

Hemolysis Measurement. The percentage hemolysis was quantitated bymeasuring the absorbance of 570 nm light by the plasma. Absorbance wasmeasured on a Hewlett-Packard 8451A spectrophotometer. The readings weretaken using cells having path lengths of approximately 0.01 cm. The 0.01cm path length was in a tape cartridge prepared as described above. Theabsorbance was converted to percent hemolysis by multiplication of theabsorbance by a conversion factor. The peak at 570 nm was used for the0.01 cm pathlength cell, and the conversion constant was 42.0.

Glass Fiber Filters. A number of glass fiber filters were tested,including GA-200 from Micro Filtration Systems (MFS), which is thefilter used in all examples unless another filter is specified. GA-200is a non-woven glass fiber filter containing glass microfibers havingtypical diameters in the range from 0.5 to 1.0 micrometer. The filter is0.70 mm thick and retained particles 2.3 μm in diameter in the liquidphase. The density of the filter is 0.25 g/cm³. Density and thicknessvalues are given prior to the slight compression that took place duringthe process of fabricating the capillary device.

Results

Blood from a patient with sickle cell anemia, blood with artificiallyproduced high and low hematocrits, and normal blood were filteredthrough the GA-200 filters to determine if blood with an abnormalhematocrit would be effectively filtered.

    ______________________________________                                        Blood Type                                                                            Filtration   Time 1* (sec)                                                                            Lysis (%)                                     ______________________________________                                        sickle cell                                                                           +            <5         0.80                                          HCT = 30                                                                              +            <5         --                                            ______________________________________                                                           Time 1*   Time 2*                                                                              Volume**                                  Blood Type                                                                              Filtration                                                                             (sec)     (sec)  (μl)                                   ______________________________________                                        Fresh blood                                                                   HCT = 48.5                                                                              +        4         12.6   2.5                                                 +        5         13     2.5                                       HCT = 33.0                                                                              +        4         8.9    5                                                   +        5         12.7   5                                         HCT = 60.0                                                                              +        4         13.2   2.5                                                 +        8         27     2.5                                                 +        7         12     2.5                                       ______________________________________                                         *Time 1 is the time between the addition of the blood to the filter and       the exiting of red blood cells from the filter. Time 2 is the time for th     blood to reach the beginning of the reagent well.                             **Volume = the volume of plasma which exited the filter before red blood      cells exited the filter.                                                 

It is evident that the filters are as effective in filtering theabnormal hematocrit blood as they are with normal blood: in fact, lowerhematocrit blood appears to flow through the filters faster than normalor high hematocrit blood.

The lower hematocrit blood was more efficiently filtered: that is, morevolume plasma per volume of blood exited the filters before the redblood cells. However, sufficient plasma was separated even in highhematocrit blood to allow plasma testing.

Comparison of Filters from MFS

A variety of filters from Micro Filtration Systems were tested for theability to filter RBCs from plasma. The nomenclature of the MFS filtersis based on their physical properties. The further along the secondletter of the name is in the alphabet, the tighter the weave of thefilter and the slower the flow through the filter. The numbers in thename correspond to the thickness of the filter: that is, the higher thenumber, the thicker the filter. Three filters from the group examinedproved satisfactory: the GA-200, two GB-100R stacked on top of eachother, and two GC-90 stacked on top of each other.

    ______________________________________                                                   Time 1  Time 2    Volume                                           Filter     (sec)   (sec)     (μl)                                                                              % Lysis*                                  ______________________________________                                        GA-200     5.0     12.8      4      0.58                                      GB-100 × 2                                                                         19      32        5      0.95                                      GC-90 × 2                                                                          --      120       5      --                                        ______________________________________                                         *Lysis measured after removal of red blood cells by centrifugation = 0.37                                                                              

Analyte Recovery After Exposure to Glass Fiber Filter

The purpose of this experiment was to determine if potential analyteswould be adsorbed by the glass fiber filter material. The analytestested were cholesterol, potassium, and total protein. The experimentwas conducted using the following protocol.

1. Serum was obtained from whole blood by drawing the blood into glassVacu-tainer tubes, transfering the blood to centrifugation tubes,letting the blood stand at room temperature for 20 minutes and thencentrifuging for 5 minutes at the blood setting on a TRIAC centrifuge(Clay Adams).

2. The sample was then split, one sample being contacted with the glassfiber filter material and the other being left alone until laboratoryanalysis.

3. The volume of the filter disks in the tape slides was 12.6 μl.Assuming 50 μl of blood is added to the filter, the ratio of bloodvolume to filter volume was approximately four. In the experiment, 2 mlof serum was contacted with a 24 mm diameter disk (depth=0.7 mm) with atotal volume of 317 μl. The blood/filter volume ratio was 2000/317=6.3in the experiment.

4. The samples containing filters were vortexed at medium speed forabout 20 seconds and then spun in a TRIAC centrifuge for 5 minutes tospin down the glass fibers. The serum was drawn off using a glass pipet.The serum was then analyzed.

    ______________________________________                                                       Without                                                                              With    Fraction                                                       filter filter  recovered                                       ______________________________________                                        CHOLESTEROL (mg/dl)                                                                            157      158     1.01                                        POTASSIUM (mEq/ml)                                                                             4.2      4.2     1.00                                        TOTAL PROTEIN (gm/dl)                                                                          7.2      7.1     0.99                                        ______________________________________                                    

The potassium, total protein, and cholesterol results indicate thatthere was almost complete recovery of these analytes after contact withthe filter.

All publications and patent applications cited in the specification areindicative of the level of skill of those skilled in the art to whichthis invention pertains. Each publication is individually hereinincorporated by reference to the same extent as if each individualpublication and patent application had been incorporated by referenceindividually in the location where cited.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious to those skilled in the art thatcertain changes and modifications may be practiced within the scope ofthe appended claims.

What is claimed is:
 1. In a clinical diagnostic device comprising ahousing having a fluid inlet port, a vented reaction area, and acapillary pathway connecting said inlet port and said reaction area andin which the driving force for the movement of liquid through saidcapillary pathway arises from capillary pressure, an improvement whichcomprises:a low-pressure filter interposed in said pathway, wherein saidfilter is selected from the group consisting of binderless glassmicrofiber filters having a particle size retention in the range fromabout 1 μm to about 3 μm, a flow path of from about 0.5 to about 0.5 mm,and a density in the range from about 0.10 to 0.30 g/cm³ and comprisesglass fibers having diameters essentially all in the range of from 0.10to 4.0 μm with at least 60% of the fibers having diameters in the rangeof from 0.10 to 1.23 μm, wherein said device contains no agglutinin forred blood cells, whereby whole blood applied to said inlet port isseparated into plasma and red blood cells by said low-pressure filter.2. The device of claim 1, wherein said filter consists essentially ofborosilicate glass fibers.
 3. The device of claim 2, wherein said filterhas a thickness of 0.50 to 0.80 nm, a particle size retention of fromabout 2.3 to 3.0 μm, and comprises glass fibers having diametersdistributed over said range of from 0.10 to 1.23 μm so that 3 or 4 equaldivisions of said range each contains a number of fibers varying by nomore than 10 number percent.